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. 2022 Feb 17;13:954. doi: 10.1038/s41467-022-28618-9

Fig. 1. Hypoxia induces macropinocytosis in HCC cells.

Fig. 1

a Immortalized liver cells MIHA, THLE3, and HCC cells MHCC97L, PLC/PRF/5, and Hep3B exposed to 21 and 1% O2 were incubated with 70 kDa tetramethylrhodamine dextran (TMR dextran) and dye-quenched FITC-conjugated BSA (DQ-FITC-BSA) for macropinosome labeling and measurement of protein degradation, respectively. Confocal images show dextran uptake/macropinosomes (red), degraded FITC-BSA (green), and nuclei (DAPI, blue). Macropinocytic indexes were calculated based on the intensities of red signals, DQ-FITC-BSA particle areas were calculated from green signals, respectively. Values were normalized to 21% O2. b MIHA, THLE3, MHCC97L, PLC/PRF/5, and Hep3B exposed to 1% O2 were treated with 50 µM EIPA or vehicle control (Ctrl). Confocal images demonstrate the macropinosome (dextran, red) and constitutively fluorescent BSA (green) within the cells. Macropinocytic indexes were calculated based on the intensities of red signals, constitutively fluorescent BSA particle areas were calculated from green signals. Values were normalized to 21% O2 Ctrl. c Left: MHCC97L cells were treated with 10 µM IPA-3 or vehicle control (Ctrl, DMSO). Confocal images show dextran uptake/macropinosomes (red) and nuclei (DAPI, blue) of MHCC97L cells exposed to 21 and 1% O2. Right: Confocal images show degraded FITC-BSA (green) and nuclei (DAPI, blue) of MHCC97L cells exposed to 21 and 1% O2. Macropinocytic indexes and DQ-FITC-BSA particle areas were normalized to 21% O2 Ctrl. d MHCC97L cells exposed to 21 and 1% O2 were treated with 200 nM lysosomal inhibitors bafilomycin A1 (BafA1), 20 μM chloroquine (CQ), or vehicle control (Ctrl, DMSO). Confocal images show dextran uptake/macropinosomes (red), degraded FITC-BSA (green), and nuclei (DAPI, blue). Macropinocytic indexes and DQ-FITC-BSA particle areas were normalized to 21% O2 Ctrl. e Confocal images demonstrate dextran uptake/macropinosomes (red), degraded DQ-FITC-BSA (green), and nuclei (DAPI, blue) of MHCC97L wild type (WT), -HIF-1α-KO#12, and -HIF-1α-KO#32 exposed to 21 and 1% O2. Macropinocytic indexes and DQ-FITC-BSA particle areas were normalized to values of 21% O2 WT. a–e Results were from 3 independent experiments. Box-and-whisker: center line, median; box limits, 25th to 75th percentiles; whiskers, min to max. Two-way ANOVA with Bonferroni correction. Source data are provided as a Source Data file.